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Carlos-Alberto Rodriguez-Barrientos, Noelia J Kunzevitzky, Alena Bartakova, Judith Zavala, Jeffrey L Goldberg, Jorge E Valdez; Optimization of Human Corneal Endothelial Cell Morphology. . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1146.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelial dysfunction is a leading cause of blindness and cell therapy approaches are being studied as an alternative to lamellar and penetrating keratoplasty. Several protocols for expanding human corneal endothelial cells (HCECs) in vitro have been developed; however, often HCECs undergo endothelial-to-mesenchymal transition and they lose their ability to form a tight, functional monolayer. Here we describe changes in HCEC morphology in the presence of various culture media supplements.
Corneas were dissected, the endothelium was peeled off and HCECs were isolated and expanded in vitro for at least 2 weeks in culture medium containing fetal bovine serum (FBS), nerve growth factor (NGF), epidermal growth factor (EGF) and bovine pituitary extract (BPE). After exhibiting morphological changes, fibroblastic HCECs were switched to basal medium supplemented with FBS and imaged daily for 7 days. Sister cultures were similarly treated and the expression of surface markers associated with HCEC morphology was evaluated by flow cytometry.
HCECs cultured in the presence of FBS and exogenous growth factors underwent endothelial to mesenchymal transition during passage 3. After switching them to a basal media supplemented only with FBS, HCECs retained the fibroblastic morphology for the first 24 hours but at 72 hours they reverted to a polygonal (canonical) shape. Increased expression of CD56+, a surface marker associated with HCEC canonical morphology, was observed 72 hours after the culture media switch.
These preliminary experiments revealed that when HCECs undergo endothelial to mesenchymal transformation, canonical morphology may be restored at least 72 hours later by switching the culture media to a stabilization media containing FBS. Future studies are aimed at analyzing the time course of this transition and may address the molecular mechanisms underlying this phenomenon
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